Familial Cancer Syndromes

• the familial occurrence of cancer

• clinical oncogenetics

• hereditary breast- en ovarian cancer

• hereditary colorectal cancer

Causes of cancer

Causes of cancer:environmentalhereditary

Hereditary factors: evidencepositive family historytwin studiesoccurrence cancer in certain ethnic

groups

History for breast cancer

Factor Relative risk High risk group

Age >10 elderly

Ethnicity 5 Western society

Age at menarche 3 <11 years of age

Age at menopauze 2 >54 years of age

Age at first full termpregnancy

3 >40 years of age

Family history >2 1st degree relative, young ageat diagnosis

Bening breast disease 4-5 Atypical hyperplasia

Carcinoma in the otherbreast

4

Socio-economical group 2 Lower socio-economical group

Diet 1.5 High intake of saturated fat

The familial occurrence of cancer

hereditary5%

familial15%

Contribution of genetic factors

sporadic~80%

Cumulative risk

Contribution of environmental factors

populationrisk

8

3

1

Relative risk

~12% to 30% to 90%

Determinants

Breast cancer:• 1 or > first degree relatives• young age (<50 years)• bilateral breast cancer or ovarian cancer• breast cancer in males

General• ‘closeness’ of relatives• age at diagnosis• ratio affected to ‘at risk’

OvCa 40j

BrCa 38j

BrCa 39j

BrCa 50j

BrCa 53j

A priori risk: moderately increased

A priori risk: highly increased

familial breast cancerhereditary breast- and ovarian cancer

Hereditary vs. familial: breast cancer

Bil BrCa 41&47j

Genetic contribution to cancer

General:twin studies: concordant vs. discordantcontribution: limitedimportant in:

colorectal cancerbreast cancerprostate cancer

Family cancer syndromes

Definition:Clinical syndrome characterized by the preferential occurrence of certain tumors in a family

often: specific tumorssometimes: associated clinical signs

Inheritance

All family cancer syndromes: segregation and therefore monogenic

all known: autosomal inheritance

most: dominantsome: recessive

most: genetic heterogeneityvariable penetrance

Autosomal dominant inheritance

1 of the parents: affectedrisk: 50%independent of genderaffected relatives in all generations

-/++/+

-/+

-/+ -/+

+/+

+/+ +/+

-/+ : carrier

+/+ : wild type

Autosomal recessive inheritance

generally no affected parentsrisk: 25%independent of genderno affected relatives

-/+ : carrier, unaffected

+/+ : wild type

-/- : affected-/++/+

-/+

+/+ -/+

-/+

-/+ -/-

The Knudson hypothesis

family cancer syndromes

Sporadic tumorsAdditional genetic aberrations

hyperplasia metaplasia dysplasia CIS

Knudson and dominant inheritance

+ -

gametogenesis

-

gametogenesis

- -+ +

Family cancer syndromes

Autosomal dominantAdenomatous polyposis APCHereditary Non-Polyposis Colorectal Cancer (HNPCC) MLH1, MSH2, MSH6Hereditary Breast- and Ovarian Cancer (HBOC) BRCA1, BRCA2Li-Fraumeni TP53, CHK2Neurofibromatosis NF1, NF2Retinoblastoma RBVon Hippel Lindau VHLWilms’ tumors WT1, 2 & 3

Autosomal recessiveAtaxia teleangiectasia ATMBloom syndrome BLMWerner’s syndrome WRNFanconi’s anemia FACCXeroderma pigmentosum XPA-E, ERCC2-5

Tumor suppressor genes

Gatekeepers:direct inhibitors van cell growthe.g.: APC

Landscapers:modulate the micro-environmente.g.: NF1

Caretakers:DNA repair genese.g.: mismatch repair genes MLH1, MSH2, MSH6

Clues to an inherited predisposition to cancer

clinical diagnosis

indication for molecular genetic

testing

genetic counselingfollow-up

risk in unaffected relatives

young age at diagnosisaggregation of rare tumorsassociation with congenital

malformationsmultiple primary tumors in an

individualbilateral tumors in paired organs

positive family historytumor typesinheritance

patternclinical

aberrations

Genetic counseling

1st counseling: intake

empirical risk estimation follow-up & preventive measures presymptomatic testing

BRCA1 or BRCA2 mutationno mutation

2nd counseling:communication of test results

mutation analysis

no indication for mutation analysis

Informed consent

Issues:• the right not to be tested • purpose of genetic testing• reliability of genetic testing• course of genetic testing• cost of genetic testing• implications of both positive and negative test results• the possibility that no additional risk information will be obtained at the completion of the test• the options for approximation of risk without genetic testing• disadvantages of genetic testing• confidentiality of the test results• possibility of discrimination• preventive measures - limited proof of efficacy• risk for carriership in children

Mutation analysis

Techniques:• PTT (protein truncation test)• DGGE (denaturing gradient gel electrophoresis)• Southern blot• PCR cDNA and gDNA• direct sequencing

Detetection ratio:• dependent of mutational spectrum• dependent of the used techniques• dependent of the a priori chance of finding a mutation in a family member

Molecular genetics testing: PTT

Forward primerGGATCC TAATACGACTCATATA GGAACAGAC CAGCATGG

Bacteriophage T7 promotortranslation initiation signal

T7

PCR

in vitro transcription/ translation

SDS - PAGE autoradiography

AUGRNA

peptide

normal length (51 kDa)

truncated peptide (30 kDa)

normal length(62 kDa)

truncated peptide (22 kDa)

T7 polymeraselysateRnase inhibitoramino acids without leucineH³ leucine

Technique of choice for the detection of nonsense mutations e.g.: BRCA1&2 exon 11

Molecular genetic testing: DGGE

GC-clamp GC-clamp

Wild type DNA (allel A) Mutant DNA (allel B)

G

C

A

T

denaturation

G

C

A

T

re-annealing

GC

AT

homoduplex DNA

allele AA

allele BB

heteroduplex DNA

G

TC

A

allele AB

allele BA

AA BB AB

HETERODUPLEXES

HOMODUPLEXES

low concentration UF

high concentration UF

technique of choice for:• substitutions• small deletions and

insertions

analogous to:SSCPHeteroduplex analysis

Presymptomatic testing

Definition

genetic testing for a known mutation in an unaffected ‘at risk’ relative

+

+

+

+++

+

-

-

+

BRCA1 Q1281X

+

Hereditary breast- and ovarian cancer

Clinical description:Breast and/or ovarian cancer:• in at least3 first degree relatives or

3 second degree relatives (in case of paternal transmission) • in at least two successive generations• in at least one patient: Dx <50 years of age

Inheritance: autosomal dominant

Genes: • BRCA1 & BRCA2 (~80%)

Epidemiology

sporadicfamilial and hereditary

15%

85%

monogenicfamilial~40%

~60%

Molecular genetics

BRCA2 BRCA1

both genes: ~800 different mutationsno ‘hotspots’, strong ‘founder effects’mutations: spread over coding sequence as well as in non coding parts

wide array of different types

Founder mutation

Linkage disequilibrium:A condition where two genes are found together in a population at a greater frequency than that predicted simply by the product of their individual gene frequencies.

Mutation that is particularly frequent in a certain population due to a common ancestor

characteristic:markers surrounding the mutation are in‘linkage disequilibrium’

Founder mutation

‘old’ ‘recent’

Founder mutation

BRCA1 IVS5+3A>G

‘Consensus value’

0,92

Wild type BRCA1 exon 5 splice donor

GTATATAAGexon 5 intron 5

Consensus splice donor sequence

GT AGTAG

AGCA

intronexon

0,53

BRCA1 IVS5+3A>G exon 5 splice donor

GTGTATAAG TATGTAAGA----

0,9122

Founder mutation BRCA1 IVS5+3A>G

Sequencing

Intron 5Exon 5

DGGE

BRCA1 & BRCA2: molecular profiling

Follow-up

Counseling• non-directive• conjointly with referring physician

Interventions• intensive screening• prophylactic surgery• chemoprophylaxis

Evidence• limited proof of efficacy• few prospective studies

Example

+

+

+

+++

+

-

-

+

Bil. BrCa 36&46j

BrCa 29j

BrCa 51jBrCa 53j

BRCA1 Q1281X

+

phenocopy

Familial breast cancer: conclusions

Hereditary breast- and ovarian cancer: • Major contribution of BRCA1 & BRCA2• Genetic testing: useful• Small group of patients with highly increased risk

Familial breast cancer:• Minor contribution of BRCA1 & BRCA2• Genetic testing: limited use• Large group of patients with moderately increased risk• Probably: genetic variants, ‘modifier genes’, multifactorial inheritance

Familial prediposition to colorectal cancer

Familial colorectal cancerPreferential occurrence of colorectal cancer in a family without evidence for a heritable cancer syndrome

Hereditary colorectal cancerHNPCC: hereditary non-polyposis colorectal carcinomaFAP: familial adenomatous polyposis

Familial Polyposis Adenomatous

Definition:familial colorectal cancer syndrome characterized by the predisposition to develop 100 to 1000 polyps

Familial Adenomatous Polyposis

Molecular Genetic Testing

FAP: autosomal dominant inheritancemutations in the APC gene

complete coding sequence

mutation analysis~90% mutations: nonsense mutationsPTT on cDNAAPC mutations in ~80% of the FAP families

Adenoma/carcinoma sequence

APC: genotype/phenotype correlations

Desmoïd tumors 10-20 % Desmoïd tumors ca. 100% Hereditary desmoid disease

CHRPE +

Attenuated FAP

Classical FAP

Attenuated FAP

NH2 COOH

3’5’

Follow-up

As soon as polyps are detected: prophylactic colectomy

HNPCC

Clinical description (Amsterdam I criteria):Colorectal cancer:• in at least 3 relatives, one of which is a first degree relative of the other two• age at diagnosis in at least one patient <50 years• in at least two successive generations• FAP is excluded

Definition:familial cancer syndrome characterized by a highly increased risk for colorectal (~80%) and endometrial cancer (~60%)

Molecular genetics

genes: • hMLH1• hMSH2• hMSH6

complete coding sequence

mutation-analyse : • DGGE• Southern Blot

Additionally MSI en immunohistochemistry

Microsatellite instability

MicrosatellitesMismatch repair

Short stretches of repititive sequences

mono-, di- and trinucleotide repeats

‘slippage’ of DNA polymerase

Microsatellite instability

Tumor:defective mismatch repair

Other tissues:normal mismatch repair

Microsatellite instability

instability microsatelliteD2S123

instability microsatelliteBAT25

B

T

B

T

Microsatellite instability

Microsatellite instability

High MSI:

prognostically favorable, even in sporadic colorectal carcinomas

Immunohistochemistry

2 hit model: absent expression of the mutant tumor suppressor in the tumor

MSSnormal MSI-H

Tumor characteristics

predeliction for the proximal colon; undifferentiated growth pattern (solid or cribriform) strong lymphocyte aggregation around the tumor

colon ascendens

colon transversum

colon descendens

MIN en CIN: gatekeepers and caretakers

MIN: microsatellite instability• mutation in caretakers• euploid• proximal colon (colon ascendens)• slow initiation, fast progression

CIN: chromosomal instability• mutation in gatekeeper• aneuploid• distal• fast initiation, slow progression

(NER: nucleotide excision repair)

Conclusions

Family cancer syndromes: small group of patients with a highly increased risk, monogenic inheritance

Familial tumors: large group of patients, moderately increased risk, probably multifactorial inheritance, considerable contribution of environmental factors

Genetic testing: especially useful in family cancer syndromes

Hereditary vs. familial: clinical distinction

Combination of different mutation analysis techniques: optimization of the mutation detection rate

Mutation analysis: laborious and time consuming

Study of familial cancer syndromes: insights into biological processes essential in the etiology and the progression of cancer and vice versa